NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.
[NMR paper] Quantifying the thermodynamics of protein unfolding using 2D NMR spectroscopy
Quantifying the thermodynamics of protein unfolding using 2D NMR spectroscopy
A topic that has attracted considerable interest in recent years is the possibility to perform thermodynamic studies of proteins directly in-cell or in complex environments which mimic the cellular interior. Nuclear magnetic resonance (NMR) could be an attractive technique for these studies but its applicability has so far been limited by technical issues. Here, we demonstrate that 2D NMR methods can be successfully applied to measure thermodynamic parameters provided that a suitable choice of...
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[NMR paper] Monitoring protein folding through high pressure NMR spectroscopy.
Monitoring protein folding through high pressure NMR spectroscopy.
Monitoring protein folding through high pressure NMR spectroscopy.
Prog Nucl Magn Reson Spectrosc. 2017 Nov;102-103:15-31
Authors: Roche J, Royer CA, Roumestand C
Abstract
High-pressure is a well-known perturbation method used to destabilize globular proteins. It is perfectly reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. In contrast to other perturbation methods such as heat or chemical denaturant that...
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11-22-2017 02:01 PM
Investigating the Structural Dynamics Transitions of Human Adipocyte Fatty Acid Binding Protein by NMR Spectroscopy
Investigating the Structural Dynamics Transitions of Human Adipocyte Fatty Acid Binding Protein by NMR Spectroscopy
Publication date: 27 January 2015
Source:Biophysical Journal, Volume 108, Issue 2, Supplement 1</br>
Author(s): Kim N. Ha , Youlin Xia , Yenchi Tran , Adedolapo Ojoawo , Gianluigi Veglia , David A. Bernlohr</br>
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[NMR paper] Monitoring fast reactions by spatially-selective and frequency-shifted continuous NMR spectroscopy: application to rapid-injection protein unfolding.
Monitoring fast reactions by spatially-selective and frequency-shifted continuous NMR spectroscopy: application to rapid-injection protein unfolding.
Related Articles Monitoring fast reactions by spatially-selective and frequency-shifted continuous NMR spectroscopy: application to rapid-injection protein unfolding.
Chem Commun (Camb). 2013 Mar 12;
Authors: Wagner GE, Sakhaii P, Bermel W, Zangger K
Abstract
The repetition rate of an NMR experiment is usually limited by the longitudinal relaxation times of the investigated molecule. Here we...
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03-14-2013 10:05 PM
Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant
Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant
September 2012
Publication year: 2012
Source:Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1818, Issue 9</br>
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Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes...
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02-03-2013 10:13 AM
[NMR paper] Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate reveal
Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.
Related Articles Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.
J Mol Biol. 2002 Sep 27;322(4):841-9
Authors: Fowler SB, Best RB, Toca Herrera JL, Rutherford TJ, Steward A, Paci E, Karplus M, Clarke J
The mechanical unfolding of an immunoglobulin domain from the...
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[NMR paper] Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven b
Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven by autolysis.
Related Articles Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven by autolysis.
FEBS Lett. 2001 May 18;497(1):59-64
Authors: Panchal SC, Bhavesh NS, Hosur RV
Structural studies in proteases have been hampered because of their inherent autolytic function. However, since autolysis is known to be mediated via protein unfolding, careful monitoring of the autolytic reaction has the potential to throw light on the...
Monitoring protein unfolding transitions by NMR-spectroscopy
Linear Mode
